text:
Things are very open with a clear explanation of your issues.
It absolutely was truly informative. Your site is very useful.
Many thanks for sharing!
Our company is a bunch of volunteers and opening a whole
new scheme in your community. Your internet site provided us with helpful
info to operate on. You've done an amazing job and our whole community will be grateful for your needs.
Hi there, I discovered your blog by means of Google as
well as looking for a comparable subject, your site came up, it is apparently great.
I've bookmarked it in my google bookmarks.
Hi there, simply changed into alert to your blog thru Google, and located that it can be truly informative.
I am just gonna watch out for brussels. I'll appreciate should
you continue this in future. Lots of folks will
probably be benefited from your writing. Cheers!
Hi! Do you know if they make any plugins to protect against hackers?
I'm kinda paranoid about losing everything I've worked hard on. Any tips?
Someone necessarily lend a hand to make critically posts I'd state.
That is the very first time I frequented your website
page and to this point? I amazed with the analysis you made to make this particular publish extraordinary.
Great process!
Asking questions are really pleasant thing if you are not understanding anything entirely, however this article provides nice understanding even.
I truly fancy appearing through a write-up that cause people to envision.
Additionally, thanks for permitting us to comment!
Here is my web site :: craigslist akron canton
I truly love your website.. Pleasant colors & theme.
Did you develop this website yourself? Please reply back
as I'm looking to create my own personal blog and would
love to know where you got this from or what the theme is
named. Cheers!
Oooo, these swans are so beautiful Rodi ! And what a wonderful place !
the water is so clear and clean and beautiful!
i don't have a passion for swans, but i see you caught some lovely duckling in you shots, too <3 love them! :D


text:
When you're dealing with a device or possibly a computer system, then you definitely can just about need to cope with files. Keep in mind that unique programs, applications or anything possess a file variety. It is like for images they are available in the widespread files like others, JPEG, PNG, GIF and BMP. When it comes to videos and movies they can are available in FLV, MKB, MP4, WMV, AVI and many additional. If those examples didn't hurt your head then there are other files for that you probably wouldn't want to know. Now of course you'll find instances any time you want to carry out converting files.
How and why you might want to convert files
- A single point is to ensure that the files will be compatible towards the device which you would use it on. Like for instance a WMV file is playable on a Windows Media Player which can be normally found on a laptop. These files won't run on a smartphone so you'll want to convert the WMV to some thing like a 3gp or MP4 file.
- In relation to converting, masterful converters use their very own ways to convert a file. These techniques typically take some time however it is accomplished effectively and efficiently.
- There are actually also applications that enable people to convert files with ease. The only factor here is that it takes time depending on the size. Like converting a WMV file the size of 3GB into a 3gp a single can take up to an hour.
Usual files getting converted
- Video and Movie files would be the usual ones that are converted. Like carrying out a super 8 film to digital and conversation.
- One particular other kind of file that individuals ordinarily convert can be a plan file. That may be of course once they would would like to run a system in a diverse device or program.
Converting files could be accomplished so long as you might have the right tools for it.

subset: commoncrawl


text:
Peugeot 2008 SUV
Price £13,615 - £20,565
- Spacious, high-quality cabin
- Economical engines
- Comfortable ride
- Dull petrol engines
- Not much fun to drive
- No four-wheel-drive option
At a glance
- Our Pick
- 1.2L PureTech 110 Allure 5dr £17,715
- The greenest
- 1.6L BlueHDi 120 S&S Allure 5dr £19,465
- The cheapest
- 1.2L PureTech 82 Access A/C 5dr £13,615
- The fastest
- 1.2L PureTech 130 Allure 5dr £18,115
- Top of the range
- 1.6L BlueHDi 120 S&S GT Line 5dr £20,565
"The Peugeot 2008 SUV is a practical rugged looking crossover with a classy interior and supermini-like running costs."
The Peugeot 2008 Crossover is a compact SUV that blends low running costs with an elevated driving position and great practicality. It goes up against cars like the Nissan Juke, Citroen C4 Cactus and Ford EcoSport, but Peugeot has deliberately designed the 2008 to look more like a high-riding estate than an SUV. The Peugeot also has a classier and more luxurious-feeling interior than its rivals.
Continuing this theme of doing things slightly differently, Peugeot doesn't offer the 2008 with four-wheel drive, but there is the option of a system called 'Grip Control'. This detects and prevents the front wheels from losing grip on slippery surfaces, including snow, sand and mud. While it's not quite as effective as a full four-wheel-drive setup, most crossover customers use their cars predominantly on the road and Grip Control should provide enough 'go-anywhere' ability for the majority of drivers.
Peugeot offers the 2008 with a 1.2-litre petrol engine or a 1.6-litre diesel, both of which are available with three different power outputs. They're all efficient, becoming even more so when fitted with Peugeot's optional 'S&S' stop-start system. The 2008 comes with either a five or six-speed manual gearbox, although the entry-level 1.2-litre PureTech 82 petrol is also available with the brand's ETG automatic transmission.
As might be expected, the diesels offer greater fuel economy – although they're about £1,500 more expensive to buy than a similarly equipped petrol model. The 74, 98 and 116bhp 1.6-litre BlueHDi diesels all return 76.3mpg and are road-tax-exempt thanks to their low CO2 emissions. The 13.8-second 0-62mph time of the 74bhp model may leave you wanting a little more oomph when overtaking on the motorway, though, so the 98bhp diesel is a little easier to live with. It's by no means rapid, though with a 0-62mph time of 12.5 seconds.
If you can stretch to it, we recommend the 116bhp diesel. That extra power means it'll go from 0-62mph in a sprightly 9.6 seconds, while it shares the excellent economy figures and road-tax exemption as the other diesels.
If you don't cover that many miles, or mainly use your car for short trips, a petrol engine should suit you better, and the 2008 is well served in this area. All of its 1.2-litre petrol engines are efficient and smooth, but we recommend the 107bhp 1.2-litre PureTech 110 S&S model. This blends strong 60.1mpg fuel economy with useable performance, going from 0-62mph in 9.9 seconds, while costing just £20 a year in road tax.
On the road, the 2008 feels safe and secure, with predictable handling – although it's not the last word in driver involvement and some may feel the small steering wheel was a curious decision on Peugeot's part, Nonetheless, the suspension is soft, body lean is well controlled in corners and this is overall a comfortable car to drive.
Despite being based on the 208 hatchback, the 2008 has enough room inside for five adults, with plenty of headroom. The boot is a reasonable if not remarkable size, though its low floor means loading heavy items is easy.
There are four trim levels. The entry-level 2008 Access is reasonably well equipped, coming with air-conditioning, driver's seat height adjustment and cruise control, while Active trim adds 16-inch alloy wheels and a seven-inch infotainment system, complete with Apple CarPlay, Bluetooth phone connectivity and a DAB radio.
We recommend the mid-range Allure model, though. This includes Grip Control (although not with the 1.2-litre PureTech 82 engine), 17-inch alloy wheels, rear parking sensors, dual-zone air-conditioning and dark-tinted rear windows, which combine to make the 2008 feel very luxurious and well equipped.
The top-spec GT Line trim includes 17-inch alloy wheels, sat nav, a reversing camera and various interior upgrades, including aluminium pedals, some black gloss trim and red stitching around the seats. At around £2,500 more than Allure trim, though, GT Line is expensive.
2008 owners are generally a happy lot, something attested to by the car's 33rd-place finish out of 200 cars in our 2015 Driver Power customer satisfaction survey. Peugeot's 10th-place finish out of 32 manufacturers also suggest build quality and reliability have improved significantly in recent years. Safety is similarly reassuring: when it was crash-tested by Euro NCAP, the Peugeot 2008 scored the full five stars.
MPG, running costs & CO2
The Peugeot 2008 achieves impressive fuel economy and emissions
Engines, drive & performance
While some versions of the Peugeot 2008 are sluggish, the 1.6-litre diesel offers good all-round performance
Interior & comfort
The Peugeot 2008 boasts a classy interior, which is more business-like than the funky Nissan Juke or Citroen C4 Cactus
Practicality & boot space
There's lots of space inside the Peugeot 2008, which, added to the practical boot, makes it an ideal car for families
Reliability & safety
Unlike older Peugeots, the Peugeot 2008 benefits from excellent reliability, and safety is top-notch, too


text:
Skip to Main Content
IEEE membership options for an individual and IEEE Xplore subscriptions for an organization offer the most affordable access to essential journal articles, conference papers, standards,
eBooks, and eLearning courses.
Learn more about:
IEEE membership
IEEE Xplore subscriptions
Software-defined radios (SDRs) will play a key role in future radio configurations because the emergence of new wireless technologies and their integration in a fourth generation of communication standards will necessitate the use of multistandard and multiband radios. SDRs use a single hardware front end but can change their frequency of operation, occupied bandwidth, and adherence to various wireless standards by calling various software algorithms. Such a solution allows inexpensive, efficient interoperability between the available standards and frequency bands.
A not-for-profit organization, IEEE is the world's largest technical professional organization dedicated to advancing technology for the benefit of humanity.© Copyright 2016 IEEE - All rights reserved. Use of this web site signifies your agreement to the terms and conditions.


text:
Sandy from above
Aerial views from the devastation of Superstorm Sandy.
Aerial views from the devastation of Superstorm Sandy.
Anthony: Love seeing the nutjob Sanders and his goon squad kicked to the curb. That fool handed Hillary her bid when he started rambling about Sweden, Denmark, and Norway. Sweden especially is in total chaos and a free fall. The rape capital of the world and overran with ISIS. Progressive fools have no brain and their bulllcheit ideology proves what an utter failure and doctrine of fools it is in real life. The GDP of the USA produced more in a month than all three of these nations combined....and the USA is a fraction of the age of these nations. They look to us for leadership not the other way around...and that is something that needs to sink into the Sander nutjobs progressives skullz.


text:
In short: Not the best race I've ever been to, could put a beginner offIn full: It was extremely cold and windy, especially running into the wind along the harbourfront and the finish got very congested with a 15 minute wait from the line to leaving. However, the medal and t-shirt were nice memento's. Definitely needed someone waiting with warm clothes as I got frozen waiting to get out of the exit queue.Date of review: March 12, 2006
In short: A good experienceIn full: A well organised / marshalled event (thankyou to everyone involved) that I will do again next yearDate of review: March 12, 2006
In short: Fast, flat course. Very well organised.In full: A very good race and well organised. There is plenty of parking space. Over twenty portaloos provided. A fairly wide starting section and a course that is one of the flattest you will come across. Good marshalling all round the course and an efficient finishing funnel. No delays in getting out of the car park at the end. Even the weather (once you got going)was about right.Well done and thanks to all those who organised and helped out at this race.Date of review: March 12, 2006
In short: Excellent race with enthusiastic spectatorsIn full: I was amazed at the number of spectators throughout the course and especially at the finish. Nice course and I smashed my PB!Date of review: March 12, 2006
In short: Great race, great fun, and another PB! Bit cold this year though!In full: Realy enjoy this race. Well organised, good course (nice and flat). Liked the music at finish area. My thanks to the organisers and marshals. See you next year.Date of review: March 12, 2006
In short: Very hilly first half, cracking finishing 2k and better organised than before at Castle AshbyIn full: I was not expecting hills like it and think that for absolute beginners this must have been a nightmare, especially the first 4 miles or so. Marshalls were effective although not desperately enthusiastic, but I suppose they had given up their Sunday for nothing and were freezing cold so fair play to them all. Ben home about 20 mins and ready for a beer.Date of review: March 12, 2006
In short: Flat and quick, book now for a new PB!!!!In full: This is a 2 lap flat, quick road race. It is fairly well organised but dont expect many trimmings. The location is a small village so facilities are limited. A good turn out for it though with a nice mix of runners and an almost certain PB time, which may explain the poularity. One for next year.Date of review: March 12, 2006
In short: Great race as ever, smashed my PB. Shame about the man in blue with the attitude but always get a few at the bigger races.Date of review: March 12, 2006
In short: Excellent race. No medals tho.In full: Excellent race. Only five pound no medals or anything at the end tho. This was my first race and I enjoyed it and would do it again next year.Date of review: March 12, 2006
In short: Really enjoyable day out.Date of review: March 12, 2006
In short: lovely setting, great organisation, friendly atmosphere and well worth turning up - thanksDate of review: March 12, 2006
In short: Good fun, knackering, faster course than usual, thanks guysDate of review: March 12, 2006
In short: Fantastic race. One of my favourite races of the year. Just one point though, can we have some sun one year?Date of review: March 11, 2006
In short: great raceDate of review: March 11, 2006
In short: Well Marshalled, Very Flat, PB Potential. Chip Timing this yearDate of review: March 11, 2006
In short: Challenging, but I like a challengeDate of review: March 11, 2006
In short: great race very friendlyIn full: very well organised,fantastic medal,and good quality teeshirt and nice touch to be presented medal by the mayor, with a back drop of the fantastic castle tamworth even on a damp cold feb morning is a great place to run and the friendly atmosphere shows the town off to be a great place to visitDate of review: March 11, 2006
In short: Good organisation; great for parking; unlucky with the weatherIn full: The organisation was first class. The details sent out with the race were an accurate representation of what actually took place. The course was safe and traffic free which was the first consideration, but we went through parts of town that I was glad that I ventured into only as part of a crowd with police and army cadets on patrol ! The hill in the middle was a bit cheeky - all you could see as you got to the bottom was a long line of runners zig zagging up the path....and every time you turned a corner there was more of it. It took the edge off the possibility of a fast time. The timing chip stuff was a bit of a puzzle with runners uncertain about whether the clock stopped as we crossed the line (...and which one? ...there were two) or when the nice man with the electronic bat waved it across your chest as we queued for the medals - the official time came up a good couple of minutes longer than my Garmin, and would have been my worst time for 3 years, except I know I was running better than that. My GPS also measured the course a bit long too. The parking facilities were great. Loads of spaces available within a short walk from the start. I only got there about 3/4 of an hour before the gun and parked no more than about 150yards away. A result. The organisers were unlucky with the weather with rain, wind and sleet right up to the off. Although the rain stopped soon thereafter, the weather remained dull and cold - not good for the older ones amongst us whose joints creak a bit when the temperature drops. In all, a very well organised race that can usefully be used as part of a larger programme of races in a season. However, not one to choose for the scenary or as an event in itself because of the location and the time of year. I'll be back. Date of review: March 11, 2006
In short: It's funny how you can only remember the hills that went up! In full: I was told that the entries had doubled this year. If this is going to be true in future too, they'll need to give some thought given to the toilet situation. An earlier start might be a good idea too. Why does it need to start at 11:30? Apart from those to whinges, it's agreat race and I'll always make it part of my racing year.Date of review: March 11, 2006
In short: pleasant start on the washlands by river trent. 2005 race was held on a nice warm day. 2 lap course, a mix of parkland and residential roadsDate of review: March 10, 2006
Visit the official Runner's World page
Follow Runner's World on Twitter
Click here
Other Natmag-Rodale Sites
Run For Charity
Injury Clinics
About Runner's World


text:
Top Definition
A dream i had last night.
I was in this large field, with a board showing charcters from animated TV shows i could kill with my massive sword. I woke up before i got to slay Emily
by anamous January 21, 2005
Trending Now
Emails are sent from email@example.com. We'll never spam you.×


text:
Le Manoir des Cèdres, Perigord - Dordogne (department)
- Free WiFi and free parking
Main amenities
- 23 smoke-free guestrooms
- Restaurant
- Breakfast available
- Seasonal outdoor pool
- Beach umbrellas
- Terrace
- Air conditioning
- Garden
- Library
- Computer station
- Front desk safe
- Meeting room
What's around
- La Roque St-Christophe (8.1 mi / 13 km)
- Grotte du Grand Roc (8.9 mi / 14.3 km)
- Cave of Lascaux (13.9 mi / 22.4 km)
- Musee National de Prehistoire (10.3 mi / 16.6 km)
- Grotte des Combarelles (11.2 mi / 18.1 km)
For families
- Cribs/infant beds (surcharge)
- Children's pool
- Playground on site
- Connecting/adjoining rooms available
- Private bathroom
- Garden
Wonderful 3 days - comfy bed - quiet location - loved having the double windows open all…
A fantastic stay. Would go back to chill and enjoy the peace. Everyone is extremely…
Le Manoir des Cèdres, Perigord - Dordogne (department)
Hotel Info: 866-539-8117from $61
- Family Room, Connecting Rooms
- Superior Triple Room
- Superior Double Room
- Comfort Double Room
- Standard Single Room
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 23 rooms
- This hotel is arranged over 2 floors
- Check-in time 4 PM-7 PM
- Check-out time is 11 AM
Required at check in
- Credit card deposit required
- Government-issued photo ID required
Travelling with others
- Pets not allowed
- Free WiFi in public areas
- Free WiFi in rooms
- Free self parking
Other information
- Smoke-free property
Nearby Three Star Perigord - Dordogne (department) hotels
In the hotel
- Buffet breakfast daily (surcharge)
- Restaurant
- Beach sun loungers
- Beach umbrellas
- Outdoor seasonal pool
- Children's pool
- Bicycle rentals on site
- Horse riding/rental on site
- Playground on site
- Golf lessons available nearby
- Golfing nearby
- Hiking/biking trails nearby
- Skydiving nearby
- Beach towels
- Pool sun loungers
- Conference space size (feet) - 430
- Conference space size (meters) - 40
- One meeting room
- Computer station
- Front desk (limited hours)
- Multilingual staff
- Number of buildings/towers - 2
- Safe-deposit box at front desk
- Garden
- Picnic area
- Terrace
- Library
- Accessible bathroom
- In-room accessibility
- Roll-in shower
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Iron/ironing board (on request)
- Blackout drapes/curtains
- Private bathroom
- Shower only
- Hair dryer
- Digital TV channels
- Desk
- Free WiFi
- Phone
- Connecting/adjoining rooms available
Top Perigord - Dordogne (department) hotels
Recently reviewed hotels in Perigord - Dordogne (department)
- O Clair de LuneFrom
"To be clear, this is an Air BnB type situation not a hotel. It is a private home with a few guest rooms rented out. That's fine but Check in only from 6 to 7:30 pm. This is not…"Jun 12, 2016
- Hôtel du PerigordFrom
"We stayed one night and was made to feel as though we were regular customers. It is on the main road through the town and it was really hot day so we had both windows open,…"Jun 10, 2016
- Comfort Hotel Régina PérigueuxFrom
"short but nice"May 31, 2016
- L'EsplanadeFrom
"This was a great find! Exactly as the picture shows - this hotel is a destination by itself. The good news: the Dordogne is full of treasures you didn't know you were looking…"May 30, 2016
Nearby hotels
Le Manoir des Cèdres, Perigord - Dordogne (department)'s small print
Also known as
- Le Manoir des Cèdres Hotel Rouffignac-Saint-Cernin-de-Reilhac
- Le Manoir des Cèdres Rouffignac-Saint-Cernin-de-Reilhac
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.
Mandatory fees
You'll be asked to pay the following charges at check in or check out:
- A tax is imposed by the city: EUR 0.90 per person, per night. This tax does not apply to children under 13 years of age.
Optional extras
Buffet breakfast is offered for an extra charge of EUR 10 for adults and EUR 4.50 for children (approximately)
Cribs/Infant beds are available for EUR 8 per night
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Recent reviews
Outstanding 4.6 /5 from 20 reviews
Very pleasant stay with very friendly and helpful staff. Great room, very quiet environment on the outskirt of a small town. Recommendations for restaurants in the vicinity were much appreciated and we were very satisfied with the experience.
Is the description of this hotel not correct? Tell us


text:
Nadine & The Citygo down to start
Resource For the City
Hot Topics Regarding Child Custody
Two parents are struggling to make sure that the future of their child will not be affected in a major way by their separation. And all this happens amid anger, disillusion and fear. It is easy to see that there are many questions that need quick answers and that parents find it hard to be friends who cooperate for the sake of their child. We have here a list of hot topics that might interest parents who are dealing with the problem of child custody.
Do both parents have the same rights when it comes to child custody and visitation?
During a divorce, the court tries to decide which change would be in the best interest of the child. Both the father and the mother have equal rights to the child's custody and both of them can visit the child following the schedule that was agreed. However, even though the parents have agreed on a certain arrangement, the court can refuse to honor it if it was determined that the child will not be favored by it.
Is the child's desire taken into consideration?
As a matter of fact, the desires of a child are very important for determining the details of the custody. A child can decide with whom he wants to live and his thoughts are carefully analyzed by the court. It is a difficult period for a child who was accustomed to live with both his father and his mother and he can be confused. Some children, trying to avoid hurting their parents, tell both parents that they want to live with them.
How will parents deal with the future disagreements?
It should not be assumed that two parents will always agree on the important things that will determine the future of their child. Even though they equally love that child, they can have different opinions and this can lead to misunderstandings. The initial parenting plan, which was agreed on during divorce, will solve these future problems. This plan explains how to deal with misunderstandings related to the future of the child. If these misunderstandings appear, sometimes the parent with whom the child lives can have the right to make the final decision, whereas in other cases, one parent is entitled to make certain decisions and the other one to make other decisions.
Can the custody decision be changed later?
A parent can decide that it is necessary to reanalyze the case and to make another decision because some major changes that can affect the child's future have appeared.
Is it a rule to offer custody to the mother?
No, both parents are considered to be equal and either of them is able to request the custody of the child. The court will analyze the case based on the evidence that is offered and the decision will be made considering the best interest of the child. Also, it is very important for the court to find out the child's preference and his opinion regarding his living with one of his parents.


text:
Contact Us
Enter Your Search:
Search tips
Search criteria
Journal titles
Results 1-25 (1068593)
« Previous
Go to page number:
Next »
Clipboard (0)
Related Articles
The Effect of Macromolecular Crowding on the Electrostatic Component of Barnase–Barstar Binding: A Computational, Implicit Solvent-Based Study
Qi, Helena W.
Nakka, Priyanka
Chen, Connie
Radhakrishnan, Mala L.
PLoS ONE
Macromolecular crowding within the cell can impact both protein folding and binding. Earlier models of cellular crowding focused on the excluded volume, entropic effect of crowding agents, which generally favors compact protein states. Recently, other effects of crowding have been explored, including enthalpically-related crowder–protein interactions and changes in solvation properties. In this work, we explore the effects of macromolecular crowding on the electrostatic desolvation and solvent-screened interaction components of protein–protein binding. Our simple model enables us to focus exclusively on the electrostatic effects of water depletion on protein binding due to crowding, providing us with the ability to systematically analyze and quantify these potentially intuitive effects. We use the barnase–barstar complex as a model system and randomly placed, uncharged spheres within implicit solvent to model crowding in an aqueous environment. On average, we find that the desolvation free energy penalties incurred by partners upon binding are lowered in a crowded environment and solvent-screened interactions are amplified. At a constant crowder density (fraction of total available volume occupied by crowders), this effect generally increases as the radius of model crowders decreases, but the strength and nature of this trend can depend on the water probe radius used to generate the molecular surface in the continuum model. In general, there is huge variation in desolvation penalties as a function of the random crowder positions. Results with explicit model crowders can be qualitatively similar to those using a lowered "effective" solvent dielectric to account for crowding, although the "best" effective dielectric constant will likely depend on multiple system properties. Taken together, this work systematically demonstrates, quantifies, and analyzes qualitative intuition-based insights into the effects of water depletion due to crowding on the electrostatic component of protein binding, and it provides an initial framework for future analyses.
Related Articles
Effects of Macromolecular Crowding on Protein Conformational Changes
Dong, Hao
Qin, Sanbo
Zhou, Huan-Xiang
PLoS Computational Biology
Many protein functions can be directly linked to conformational changes. Inside cells, the equilibria and transition rates between different conformations may be affected by macromolecular crowding. We have recently developed a new approach for modeling crowding effects, which enables an atomistic representation of "test" proteins. Here this approach is applied to study how crowding affects the equilibria and transition rates between open and closed conformations of seven proteins: yeast protein disulfide isomerase (yPDI), adenylate kinase (AdK), orotidine phosphate decarboxylase (ODCase), Trp repressor (TrpR), hemoglobin, DNA β-glucosyltransferase, and Ap4A hydrolase. For each protein, molecular dynamics simulations of the open and closed states are separately run. Representative open and closed conformations are then used to calculate the crowding-induced changes in chemical potential for the two states. The difference in chemical-potential change between the two states finally predicts the effects of crowding on the population ratio of the two states. Crowding is found to reduce the open population to various extents. In the presence of crowders with a 15 Å radius and occupying 35% of volume, the open-to-closed population ratios of yPDI, AdK, ODCase and TrpR are reduced by 79%, 78%, 62% and 55%, respectively. The reductions for the remaining three proteins are 20–44%. As expected, the four proteins experiencing the stronger crowding effects are those with larger conformational changes between open and closed states (e.g., as measured by the change in radius of gyration). Larger proteins also tend to experience stronger crowding effects than smaller ones [e.g., comparing yPDI (480 residues) and TrpR (98 residues)]. The potentials of mean force along the open-closed reaction coordinate of apo and ligand-bound ODCase are altered by crowding, suggesting that transition rates are also affected. These quantitative results and qualitative trends will serve as valuable guides for expected crowding effects on protein conformation changes inside cells.
Author Summary
The biophysical properties of proteins inside cells can be expected to be quite different from those typically measured by in vitro experiments in dilute solutions. In particular, intracellular macromolecular crowding may significantly affect the equilibria and transition rates between different conformations of a protein, and hence its functions. What are the trends and magnitudes of such crowding effects? We address this question here by applying a recently developed approach for modeling crowding. Seven proteins, each with structures for both an open state and a closed state, are studied. Crowding exerts significant effects on the open-closed equilibria of four proteins and more modest effects on the remaining three. Potentials of mean force along the open-closed reaction coordinate, and hence transition rates, are similarly affected. The extent of conformational changes is the main determinant for the magnitudes of crowding effects, but the protein size also plays an important role. The effects of crowding become stronger as the protein size increases. Conformational transitions of the ribosome, an extremely large complex, during translation are predicted to experience particularly strong effects of intracellular crowding. We conclude that deduction of intracellular behaviors from in vitro experiments requires explicit consideration of crowding effects.
Related Articles
Macromolecular Crowding as a Suppressor of Human IAPP Fibril Formation and Cytotoxicity
Seeliger, Janine
Werkmüller, Alexander
Winter, Roland
PLoS ONE
The biological cell is known to exhibit a highly crowded milieu, which significantly influences protein aggregation and association processes. As several cell degenerative diseases are related to the self-association and fibrillation of amyloidogenic peptides, understanding of the impact of macromolecular crowding on these processes is of high biomedical importance. It is further of particular relevance as most in vitro studies on amyloid aggregation have been performed in diluted solution which does not reflect the complexity of their cellular surrounding. The study presented here focuses on the self-association of the type-2 diabetes mellitus related human islet amyloid polypeptide (hIAPP) in various crowded environments including network-forming macromolecular crowding reagents and protein crowders. It was possible to identify two competing processes: a crowder concentration and type dependent stabilization of globular off-pathway species and a – consequently - retarded or even inhibited hIAPP fibrillation reaction. The cause of these crowding effects was revealed to be mainly excluded volume in the polymeric crowders, whereas non-specific interactions seem to be most dominant in protein crowded environments. Specific hIAPP cytotoxicity assays on pancreatic β-cells reveal non-toxicity for the stabilized globular species, in contrast to the high cytotoxicity imposed by the normal fibrillation pathway. From these findings it can be concluded that cellular crowding is able to effectively stabilize the monomeric conformation of hIAPP, hence enabling the conduction of its normal physiological function and prevent this highly amyloidogenic peptide from cytotoxic aggregation and fibrillation.
Related Articles
An FFT-based method for modeling protein folding and binding under crowding: benchmarking on ellipsoidal and all-atom crowders
Qin, Sanbo
Zhou, Huan-Xiang
Journal of chemical theory and computation
It is now well recognized that macromolecular crowding can exert significant effects on protein folding and binding stability. In order to calculate such effects in direct simulations of proteins mixed with bystander macromolecules, the latter (referred to as crowders) are usually modeled as spheres and the proteins represented at a coarse-grained level. Our recently developed postprocessing approach allows the proteins to be represented at the all-atom level but, for computational efficiency, has only been implemented for spherical crowders. Modeling crowder molecules in cellular environments and in vitro experiments as spheres may distort their effects on protein stability. Here we present a new method that is capable for treating aspherical crowders. The idea, borrowed from protein-protein docking, is to calculate the excess chemical potential of the proteins in crowded solution by fast Fourier transform (FFT). As the first application, we studied the effects of ellipsoidal crowders on the folding and binding free energies of all-atom proteins, and found, in agreement with previous direct simulations with coarse-grained protein models, that the aspherical crowders exert greater stabilization effects than spherical crowders of the same volume. Moreover, as demonstrated here, the FFT-based method has the important property that its computational cost does not increase strongly even when the level of details in representing the crowders is increased all the way to all-atom, thus significantly accelerating realistic modeling of protein folding and binding in cell-like environments.
Related Articles
Volume Exclusion and Soft Interaction Effects on Protein Stability under Crowded Conditions†
Miklos, Andrew C.
Li, Conggang
Sharaf, Naima G.
Pielak, Gary J.
Most proteins function in nature under crowded conditions, and crowding can change protein properties. Quantification of crowding effects, however, is difficult because solutions containing hundreds of grams per liter of macromolecules often interfere with observing the protein being studied. Models for macromolecular crowding tend to focus on the steric effects of crowders, neglecting potential chemical interactions between the crowder and the test protein. Here, we report the first systematic, quantitative, residue-level study of crowding effects on the equilibrium stability of a globular protein. We used a system comprising poly(vinylpyrrolidone)s (PVPs) of varying molecular weights as crowding agents and chymotrypsin inhibitor 2 (CI2) as a small globular test protein. Stability was quantified with NMR-detected amide 1H exchange. We analyze the data in terms of hard particle exclusion, confinement, and soft interactions. For all crowded conditions, nearly every observed residue experiences a stabilizing effect. The exceptions are residues where stabilities are unchanged. At a PVP concentration of 100 g/L, the data are consistent with theories of hard particle exclusion. At higher concentrations, the data are more consistent with confinement. The data show that the crowder also stabilizes the test protein by weakly binding its native state. We conclude that the role of native-state binding and other soft interactions need to be seriously considered when applying both theory and experiment to studies of macromolecular crowding.
Related Articles
Conformational Sampling of Peptides in the Presence of Protein Crowders from AA/CG-Multiscale Simulations
Predeus, Alexander V.
Gul, Seref
Gopal, Srinivasa M.
Feig, Michael
The Journal of Physical Chemistry. B
Macromolecular crowding is recognized as an important factor influencing folding and conformational dynamics of proteins and nucleic acids. Previous views of crowding have focused on the mostly entropic volume exclusion effect of crowding but recent studies are indicating the importance of enthalpic effects, in particular changes in electrostatic interactions due to crowding. Here, temperature replica exchange molecular dynamics simulations of trp-cage and melittin in the presence of explicit protein crowders are presented to further examine the effect of protein crowders on peptide dynamics. The simulations involve a three-component multiscale modeling scheme where the peptides are represented at atomistic level, the crowder proteins at a coarse-grained level, and the surrounding aqueous solvent as implicit solvent. This scheme optimally balances a physically realistic description for the peptide with computational efficiency. The multiscale simulations were compared with simulations of the same peptides in different dielectric environments with dielectric constants ranging from 5 to 80. It is found that the sampling in the presence of the crowders resembles sampling with reduced dielectric constants between 10 and 40. Furthermore, diverse conformational ensembles are generated in the presence of crowders including partially unfolded states for trp-cage. These findings emphasize the importance of enthalpic interactions over volume exclusion effects in describing the effects of cellular crowding.
protein-protein interactions; molecular dynamics; coarse-graining; multiscale simulations; replica exchange; crowding
Related Articles
Macromolecular crowding remodels the energy landscape of a protein by favoring a more compact unfolded state
Hong, Jiang
Gierasch, Lila M.
Journal of the American Chemical Society
The interior of cells is highly crowded with macromolecules, which impacts all physiological processes. To explore how macromolecular crowding may influence cellular protein folding, we interrogated the folding landscape of a model β-rich protein, cellular retinoic acid-binding protein I (CRABP I), in the presence of an inert crowding agent (Ficoll 70). Urea titrations revealed a crowding-induced change in the water-accessible polar amide surface of its denatured state, based on an observed ca. 15% decrease in the m-value (the change in unfolding free energy with respect to urea concentration), and the effect of crowding on the equilibrium stability of CRABP I was less than our experimental error (i.e., ≤1.2 kcal/mol). Consequently, we directly probed the effect of crowding on the denatured state of CRABP I by measuring side chain accessibility using iodide quenching of tryptophan fluorescence and chemical modification of cysteines. We observed that the urea-denatured state is more compact under crowded conditions, and the observed extent of reduction of the m value by crowding agent is fully consistent with the extent of reduction of the accessibility of the Trp and Cys probes, suggesting a random and nonspecific compaction of the unfolded state. The thermodynamic consequences of crowding-induced compaction are discussed. In addition, over a wide range of Ficoll concentration, crowding significantly retarded the unfolding kinetics of CRABP I without influencing the urea dependence of the unfolding rate, arguing for no appreciable change in the nature of the transition state. Our results demonstrate how macromolecular crowding may influence protein folding by effects both on the unfolded state ensemble and on unfolding kinetics. (end of abstract)
Related Articles
Effects of Macromolecular Crowding on the Conformational Ensembles of Disordered Proteins
Qin, Sanbo
Zhou, Huan-Xiang
The journal of physical chemistry letters
Due to their conformational malleability, intrinsically disordered proteins (IDPs) are particularly susceptible to influences of crowded cellular environments. Here we report a computational study of the effects of macromolecular crowding on the conformational ensemble of a coarse-grained IDP model, by using two approaches. In one, the IDP is simulated along with the crowders; in the other, crowder-free simulations are postprocessed to predict the conformational ensembles under crowding. We found significant decreases in the radius of gyration of the IDP under crowding, and suggest repulsive interactions with crowders as a common cause for chain compaction in a number of experimental studies. The postprocessing approach accurately reproduced the conformational ensembles of the IDP in the direct simulations here, and holds enormous potential for realistic modeling of IDPs under crowding, by permitting thorough conformation sampling for the proteins even when they and the crowders are both represented at the all-atom level.
Intrinsically disordered proteins; macromolecular crowding; postprocessing approach
Related Articles
A Didactic Model of Macromolecular Crowding Effects on Protein Folding
Tsao, Douglas
Minton, Allen P.
Dokholyan, Nikolay V.
PLoS ONE
A didactic model is presented to illustrate how the effect of macromolecular crowding on protein folding and association is modeled using current analytical theory and discrete molecular dynamics. While analytical treatments of crowding may consider the effect as a potential of average force acting to compress a polypeptide chain into a compact state, the use of simulations enables the presence of crowding reagents to be treated explicitly. Using an analytically solvable toy model for protein folding, an approximate statistical thermodynamic method is directly compared to simulation in order to gauge the effectiveness of current analytical crowding descriptions. Both methodologies are in quantitative agreement under most conditions, indication that both current theory and simulation methods are capable of recapitulating aspects of protein folding even by utilizing a simplistic protein model.
Related Articles
Reduced native state stability in crowded cellular environment due to protein-protein interactions
Harada, Ryuhei
Tochio, Naoya
Kigawa, Takanori
Sugita, Yuji
Feig, Michael
Journal of the American Chemical Society
The effect of cellular crowding environments on protein structure and stability is a key issue in molecular and cellular biology. The classical view of crowding emphasizes the volume exclusion effect that generally favors compact, native states. Here, results from molecular dynamics simulations and NMR experiments show that protein crowders may destabilize native states via protein-protein interactions. In the model system considered here, mixtures of villin head piece and protein G at high concentrations, villin structures become increasingly destabilized upon increasing crowder concentrations. The denatured states observed in the simulation involve partial unfolding as well as more subtle conformational shifts. The unfolded states remain overall compact and only partially overlap with unfolded ensembles at high temperature and in the presence of urea. NMR measurements on the same systems confirm structural changes upon crowding based on changes of chemical shifts relative to dilute conditions. An analysis of protein-protein interactions and energetic aspects suggests the importance of enthalpic and solvation contributions to the crowding free energies that challenge an entropic-centered view of crowding effects.
Molecular dynamics simulation; folding; villin; protein-protein interactions
Related Articles
A method for computing association rate constants of atomistically represented proteins under macromolecular crowding
Qin, Sanbo
Cai, Lu
Zhou, Huan-Xiang
Physical biology
In cellular environments, two protein molecules on their way to form a specific complex encounter many bystander macromolecules. The latter molecules, or crowders, affect both the energetics of the interaction between the test molecules and the dynamics of their relative motion. In earlier work (Zhou and Szabo 1991 J. Chem. Phys. 95 5948-52), it has been shown that, in modeling the association kinetics of the test molecules, the presence of crowders can be accounted for by their energetic and dynamic effects. The recent development of the transient-complex theory for protein association in dilute solutions makes it possible to easily incorporate the energetic and dynamic effects of crowders. The transient complex refers to a late on-pathway intermediate, in which the two protein molecules have near-native relative separation and orientation but have yet to form the many short-range specific interactions of the native complex. The transient-complex theory predicts the association rate constant as ka = ka0exp (−ΔGel*/kBT), where ka0 is the "basal" rate constant for reaching the transient complex by unbiased diffusion, and the Boltzmann factors captures the influence of long-range electrostatic interactions between the protein molecules. Crowders slow down the diffusion, therefore reducing the basal rate constant (to kac0), and induce an effective interaction energy ΔGc. We show that the latter interaction energy for atomistic proteins in the presence of spherical crowders is "long"-ranged, allowing the association rate constant under crowding to be computed as kac = kac0exp[−(ΔGel* + ΔGc*)/kBT]. Applications demonstrate that this computational method allows for realistic modeling of protein association kinetics under crowding.
association rate constant; transient complex; macromolecular crowding; diffusion; electrostatic rate enhancement
Related Articles
Effects of Molecular Crowding on the Dynamics of Intrinsically Disordered Proteins
Cino, Elio A.
Karttunen, Mikko
Choy, Wing-Yiu
PLoS ONE
Inside cells, the concentration of macromolecules can reach up to 400 g/L. In such crowded environments, proteins are expected to behave differently than in vitro. It has been shown that the stability and the folding rate of a globular protein can be altered by the excluded volume effect produced by a high density of macromolecules. However, macromolecular crowding effects on intrinsically disordered proteins (IDPs) are less explored. These proteins can be extremely dynamic and potentially sample a wide ensemble of conformations under non-denaturing conditions. The dynamic properties of IDPs are intimately related to the timescale of conformational exchange within the ensemble, which govern target recognition and how these proteins function. In this work, we investigated the macromolecular crowding effects on the dynamics of several IDPs by measuring the NMR spin relaxation parameters of three disordered proteins (ProTα, TC1, and α-synuclein) with different extents of residual structures. To aid the interpretation of experimental results, we also performed an MD simulation of ProTα. Based on the MD analysis, a simple model to correlate the observed changes in relaxation rates to the alteration in protein motions under crowding conditions was proposed. Our results show that 1) IDPs remain at least partially disordered despite the presence of high concentration of other macromolecules, 2) the crowded environment has differential effects on the conformational propensity of distinct regions of an IDP, which may lead to selective stabilization of certain target-binding motifs, and 3) the segmental motions of IDPs on the nanosecond timescale are retained under crowded conditions. These findings strongly suggest that IDPs function as dynamic structural ensembles in cellular environments.
Related Articles
Molecular Crowding Favors Reactivity of a Human Ribozyme Under Physiological Ionic Conditions
Strulson, Christopher A.
Yennawar, Neela H.
Rambo, Robert P.
Bevilacqua, Philip C.
In an effort to relate RNA folding to function under cellular-like conditions, we monitored the self-cleavage reaction of the human hepatitis delta virus (HDV)-like CPEB3 ribozyme in the background of physiological ionic concentrations and various crowding and cosolute agents. We found that under physiological free Mg2+ concentrations (~0.1 to 0.5 mM Mg2+), both crowders and cosolutes stimulate the rate of self-cleavage, up to ~6-fold, but that in 10 mM Mg2+—conditions widely used for in vitro ribozyme studies—these same additives have virtually no effect on self-cleavage rate. We further observe a dependence of self-cleavage rate on crowder size, wherein rate stimulation is diminished for crowders larger than the size of the unfolded RNA. Monitoring effects of crowding and cosolute agents on rates in biological amounts of urea revealed additive-promoted increases in both low and high Mg2+ concentrations, with a maximal stimulation of more than 10-fold and a rescue of the rate to its urea-free values. Small-angle X-ray scattering (SAXS) experiments reveal a structural basis for this stimulation in that higher molecular weight crowding agents favor a more compact form of the ribozyme in 0.5 mM Mg2+ that is essentially equivalent to the form under standard ribozyme conditions of 10 mM Mg2+ and no crowder. This finding suggests that at least a portion of the rate enhancement arises from favoring the native RNA tertiary structure. We conclude that cellular-like crowding supports ribozyme reactivity by favoring a compact form of the ribozyme, but only under physiological ionic and cosolute conditions.
Related Articles
Modulation of Calmodulin Plasticity by the Effect of Macromolecular Crowding
Homouz, Dirar
Sanabria, Hugo
Waxham, M. Neal
Cheung, Margaret S.
Journal of molecular biology
In vitro biochemical reactions are most often studied in dilute solution, a poor mimic of the intracellular space of eukaryotic cells which are crowded with mobile and immobile macromolecules. Such crowded conditions exert volume exclusion and other entropic forces that have the potential to impact chemical equilibria and reaction rates. In this article, we used the well characterized and ubiquitous molecule calmodulin (CaM) and a combination of theoretical and experimental approaches to address how crowding impacts CaM's conformational plasticity. CaM is a dumbbell shaped molecule that contains four EF hands (two in the N-lobe and two in the C-lobe) that each could bind Ca2+ leading to stabilization of certain substates that favor interactions with other target proteins. Using coarse-grained molecular simulations, we explored the distribution of CaM conformations in the presence of crowding agents. These predictions in which crowding effects enhances the population of compact structures were then confirmed in experimental measurements using fluorescence resonance energy transfer techniques of donor/acceptor labeled CaM under normal and crowded conditions. We further explored the folding energy landscape and examined the structural characteristics of CaM at free energy basins using protein reconstruction methods. We discovered that crowding stabilizes several different compact conformations, which reflects the inherent plasticity in CaM's structure. From these results, we suggest that the EF hands in the C-lobe are flexible and can be thought of as a switch, while those in the N-lobe are stiff as analogous to a rheostat. New combinatorial signaling properties may arise from the product of the differential plasticity of the two distinct lobes of CaM in the presence of crowding. We discuss the implications of these results for modulating CaM's ability to bind Ca2+ and target proteins.
Calmodulin; EF-hands; macromolecular crowding; coarse-grained molecular simulations; FRET; Ca2+-signaling
Related Articles
Simulation and Modeling of Crowding Effects on the Thermodynamic and Kinetic Properties of Proteins with Atomic Details
Zhou, Huan-Xiang
Qin, Sanbo
Biophysical reviews
Recent experimental studies of protein folding and binding under crowded solutions suggest that crowding agents exert subtle influences on the thermodynamic and kinetic properties of the proteins. While some of the crowding effects can be understood qualitatively from simple models of the proteins, quantitative rationalization of these effects requires an atomistic representation of the protein molecules in modeling their interactions with crowders. A computational approach, known as postprocessing, has opened the door for atomistic modeling of crowding effects. This review summarizes the applications of the postprocessing approach for studying crowding effects on the thermodynamics and kinetics of protein folding, conformational transition, and binding. The integration of atomistic modeling with experiments in crowded solutions promises new insight into biochemical processes in cellular environments.
macromolecular crowding; protein folding; protein binding; simulation; modeling; postprocessing
Related Articles
Protein Folding Requires Crowd Control in a Simulated Cell
Jefferys, Benjamin R.
Kelley, Lawrence A.
Sternberg, Michael J.E.
Journal of Molecular Biology
Macromolecular crowding has a profound effect upon biochemical processes in the cell. We have computationally studied the effect of crowding upon protein folding for 12 small domains in a simulated cell using a coarse-grained protein model, which is based upon Langevin dynamics, designed to unify the often disjoint goals of protein folding simulation and structure prediction. The model can make predictions of native conformation with accuracy comparable with that of the best current template-free models. It is fast enough to enable a more extensive analysis of crowding than previously attempted, studying several proteins at many crowding levels and further random repetitions designed to more closely approximate the ensemble of conformations. We found that when crowding approaches 40% excluded volume, the maximum level found in the cell, proteins fold to fewer native-like states. Notably, when crowding is increased beyond this level, there is a sudden failure of protein folding: proteins fix upon a structure more quickly and become trapped in extended conformations. These results suggest that the ability of small protein domains to fold without the help of chaperones may be an important factor in limiting the degree of macromolecular crowding in the cell. Here, we discuss the possible implications regarding the relationship between protein expression level, protein size, chaperone activity and aggregation.
TM, template modelling; macromolecular crowding; protein structure prediction; protein misfolding; protein aggregation; protein expression
Related Articles
Cell-Free Protein Expression under Macromolecular Crowding Conditions
Ge, Xumeng
Luo, Dan
Xu, Jianfeng
PLoS ONE
Cell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process.
Methodology/Principal Findings
Using cell-free synthesis of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments.
Three macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE.
Related Articles
An upper limit for macromolecular crowding effects
Miklos, Andrew C
Li, Conggang
Sorrell, Courtney D
Lyon, L Andrew
Pielak, Gary J
BMC Biophysics
Solutions containing high macromolecule concentrations are predicted to affect a number of protein properties compared to those properties in dilute solution. In cells, these macromolecular crowders have a large range of sizes and can occupy 30% or more of the available volume. We chose to study the stability and ps-ns internal dynamics of a globular protein whose radius is ~2 nm when crowded by a synthetic microgel composed of poly(N-isopropylacrylamide-co-acrylic acid) with particle radii of ~300 nm.
Our studies revealed no change in protein rotational or ps-ns backbone dynamics and only mild (~0.5 kcal/mol at 37°C, pH 5.4) stabilization at a volume occupancy of 70%, which approaches the occupancy of closely packing spheres. The lack of change in rotational dynamics indicates the absence of strong crowder-protein interactions.
Our observations are explained by the large size discrepancy between the protein and crowders and by the internal structure of the microgels, which provide interstitial spaces and internal pores where the protein can exist in a dilute solution-like environment. In summary, microgels that interact weakly with proteins do not strongly influence protein dynamics or stability because these large microgels constitute an upper size limit on crowding effects.
Related Articles
Influence of Nanoparticle Size and Shape on Oligomer Formation of an Amyloidogenic Peptide
O'Brien, Edward P.
Straub, John. E.
Brooks, Bernard R.
Thirumalai, D.
The journal of physical chemistry letters
Understanding the influence of macromolecular crowding and nanoparticles on the formation of in-register β-sheets, the primary structural component of amyloid fibrils, is a first step towards describing in vivo protein aggregation and interactions between synthetic materials and proteins. Using all atom molecular simulations in implicit solvent we illustrate the effects of nanoparticle size, shape, and volume fraction on oligomer formation of an amyloidogenic peptide from the transthyretin protein. Surprisingly, we find that inert spherical crowding particles destabilize in-register β-sheets formed by dimers while stabilizing β-sheets comprised of trimers and tetramers. As the radius of the nanoparticle increases crowding effects decrease, implying smaller crowding particles have the largest influence on the earliest amyloid species. We explain these results using a theory based on the depletion effect. Finally, we show that spherocylindrical crowders destabilize the ordered β-sheet dimer to a greater extent than spherical crowders, which underscores the influence of nanoparticle shape on protein aggregation.
crowding; in vivo; early events; amyloid
Related Articles
Structure of Metaphase Chromosomes: A Role for Effects of Macromolecular Crowding
Hancock, Ronald
PLoS ONE
In metaphase chromosomes, chromatin is compacted to a concentration of several hundred mg/ml by mechanisms which remain elusive. Effects mediated by the ionic environment are considered most frequently because mono- and di-valent cations cause polynucleosome chains to form compact ∼30-nm diameter fibres in vitro, but this conformation is not detected in chromosomes in situ. A further unconsidered factor is predicted to influence the compaction of chromosomes, namely the forces which arise from crowding by macromolecules in the surrounding cytoplasm whose measured concentration is 100–200 mg/ml. To mimic these conditions, chromosomes were released from mitotic CHO cells in solutions containing an inert volume-occupying macromolecule (8 kDa polyethylene glycol, 10.5 kDa dextran, or 70 kDa Ficoll) in 100 µM K-Hepes buffer, with contaminating cations at only low micromolar concentrations. Optical and electron microscopy showed that these chromosomes conserved their characteristic structure and compaction, and their volume varied inversely with the concentration of a crowding macromolecule. They showed a canonical nucleosomal structure and contained the characteristic proteins topoisomerase IIα and the condensin subunit SMC2. These observations, together with evidence that the cytoplasm is crowded in vivo, suggest that macromolecular crowding effects should be considered a significant and perhaps major factor in compacting chromosomes. This model may explain why ∼30-nm fibres characteristic of cation-mediated compaction are not seen in chromosomes in situ. Considering that crowding by cytoplasmic macromolecules maintains the compaction of bacterial chromosomes and has been proposed to form the liquid crystalline chromosomes of dinoflagellates, a crowded environment may be an essential characteristic of all genomes.
Related Articles
Computer Simulations of the Bacterial Cytoplasm
Frembgen-Kesner, Tamara
Elcock, Adrian H.
Biophysical reviews
Ever since the pioneering work of Minton, it has been recognized that the highly crowded interior of biological cells has the potential to cause dramatic changes to both the kinetics and thermodynamics of protein folding and association events relative to behavior that might be observed in dilute solution conditions. One very productive way to explore the effects of crowding on protein behavior has been to use macromolecular crowding agents that exclude volume without otherwise strongly interacting with the protein under study. An alternative, complementary approach to understanding the potential differences between behavior in vivo and in vitro is to develop simulation models that explicitly attempt to model intracellular environments at the molecular scale, and that thereby can be used to directly monitor biophysical behavior in conditions that accurately mimic those encountered in vivo. It is with studies of this type that the present review will be concerned. We review in detail four published studies that have attempted to simulate the structure and dynamics of the bacterial cytoplasm and that have each explored different biophysical aspects of the cellular interior. While each of these studies has yielded important new insights, there are important questions that remain to be resolved in terms of determining the relative contributions made by energetic and hydrodynamic interactions to the diffusive behavior of macromolecules and to the thermodynamics of protein folding and associations in vivo. Some possible new directions for future generation simulation models of the cytoplasm are outlined.
computer simulation; bacterial cytoplasm; macromolecular crowding; energetic interactions; hydrodynamic interactions
Related Articles
A Generalized Fundamental Measure Theory for Atomistic Modeling of Macromolecular Crowding
Qin, Sanbo
Zhou, Huan-Xiang
Physical review. E, Statistical, nonlinear, and soft matter physics
2010;81(3 Pt 1):031919.
Macromolecular crowding inside cells affects the thermodynamic and kinetic properties of proteins. The scaled particle theory (SPT) has played an important role toward establishing a qualitative picture for the effects of crowding. However, SPT-based modeling lacks molecular details. Molecular dynamics simulations overcome this limitation, but at great computational cost. Here we present a theoretical method for modeling crowding at the atomic level. The method makes it possible to achieve exhaustive conformational sampling in modeling crowding effects and to tackle challenges posed by large protein oligomers and by complex mixtures of crowders.
Related Articles
Diffusion, Crowding & Protein Stability in a Dynamic Molecular Model of the Bacterial Cytoplasm
McGuffee, Sean R.
Elcock, Adrian H.
PLoS Computational Biology
A longstanding question in molecular biology is the extent to which the behavior of macromolecules observed in vitro accurately reflects their behavior in vivo. A number of sophisticated experimental techniques now allow the behavior of individual types of macromolecule to be studied directly in vivo; none, however, allow a wide range of molecule types to be observed simultaneously. In order to tackle this issue we have adopted a computational perspective, and, having selected the model prokaryote Escherichia coli as a test system, have assembled an atomically detailed model of its cytoplasmic environment that includes 50 of the most abundant types of macromolecules at experimentally measured concentrations. Brownian dynamics (BD) simulations of the cytoplasm model have been calibrated to reproduce the translational diffusion coefficients of Green Fluorescent Protein (GFP) observed in vivo, and "snapshots" of the simulation trajectories have been used to compute the cytoplasm's effects on the thermodynamics of protein folding, association and aggregation events. The simulation model successfully describes the relative thermodynamic stabilities of proteins measured in E. coli, and shows that effects additional to the commonly cited "crowding" effect must be included in attempts to understand macromolecular behavior in vivo.
Author Summary
The interior of a typical bacterial cell is a highly crowded place in which molecules must jostle and compete with each other in order to carry out their biological functions. The conditions under which such molecules are typically studied in vitro, however, are usually quite different: one or a few different types of molecules are studied as they freely diffuse in a dilute, aqueous solution. There is therefore a significant disconnect between the conditions under which molecules can be most usefully studied and the conditions under which such molecules usually "live", and developing ways to bridge this gap is likely to be important for properly understanding molecular behavior in vivo. Toward this end, we show in this work that computer simulations can be used to model the interior of bacterial cells at a near atomic level of detail: the rates of diffusion of proteins are matched to known experimental values, and their thermodynamic stabilities are found to be in good agreement with the few measurements that have so far been performed in vivo. While the simulation approach is certainly not free of assumptions, it offers a potentially important complement to experimental techniques and provides a vivid illustration of molecular behavior inside a biological cell that is likely to be of significant educational value.
Related Articles
CaMKII Binding to GluN2B Is Differentially Affected by Macromolecular Crowding Reagents
Goodell, Dayton J.
Eliseeva, Tatiana A.
Coultrap, Steven J.
Bayer, K. Ulrich
PLoS ONE
Binding of the Ca2+/calmodulin(CaM)-dependent protein kinase II (CaMKII) to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B controls long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. Regulation of this interaction is well-studied biochemically, but not under conditions that mimic the macromolecular crowding found within cells. Notably, previous molecular crowding experiments with lysozyme indicated an effect on the CaMKII holoenzyme conformation. Here, we found that the effect of molecular crowding on Ca2+/CaM-induced CaMKII binding to immobilized GluN2B in vitro depended on the specific crowding reagent. While binding was reduced by lysozyme, it was enhanced by BSA. The ATP content in the BSA preparation caused CaMKII autophosphorylation at T286 during the binding reaction; however, enhanced binding was also observed when autophosphorylation was blocked. Importantly, the positive regulation by nucleotide and BSA (as well as other macromolecular crowding reagents) did not alleviate the requirement for CaMKII stimulation to induce GluN2B binding. The differential effect of lysozyme (14 kDa) and BSA (66 kDa) was not due to size difference, as both dextran-10 and dextran-70 enhanced binding. By contrast, crowding with immunoglobulin G (IgG) reduced binding. Notably, lysozyme and IgG but not BSA directly bound to Ca2+/CaM in an overlay assay, suggesting a competition of lysozyme and IgG with the Ca2+/CaM-stimulus that induces CaMKII/GluN2B binding. However, lysozyme negatively regulated binding even when it was instead induced by CaMKII T286 phosphorylation. Alternative modes of competition would be with CaMKII or GluN2B, and the negative effects of lysozyme and IgG indeed also correlated with specific or non-specific binding to the immobilized GluN2B. Thus, the effect of any specific crowding reagent can differ, depending on its additional direct effects on CaMKII/GluN2B binding. However, the results of this study also indicate that, in principle, macromolecular crowding enhances CaMKII binding to GluN2B.
Related Articles
Folding, Stability and Shape of Proteins in Crowded Environments: Experimental and Computational Approaches
Samiotakis, Antonios
Wittung-Stafshede, Pernilla
Cheung, Margaret S.
International Journal of Molecular Sciences
How the crowded environment inside cells affects folding, stability and structures of proteins is a vital question, since most proteins are made and function inside cells. Here we describe how crowded conditions can be created in vitro and in silico and how we have used this to probe effects on protein properties. We have found that folded forms of proteins become more compact in the presence of macromolecular crowding agents; if the protein is aspherical, the shape also changes (extent dictated by native-state stability and chemical conditions). It was also discovered that the shape of the macromolecular crowding agent modulates the folding mechanism of a protein; in addition, the extent of asphericity of the protein itself is an important factor in defining its folding speed.
Macromolecular crowding; Ficoll® 70; energy landscape theory; off-lattice model; excluded volume effect; protein folding mechanism; spectroscopy
Related Articles
Results 1-25 (1068593)
« Previous
Go to page number:
Next »
Remove citation from clipboard
Add citation to clipboard
This will clear all selections from your clipboard. Do you wish proceed?
Clipboard is full! Please remove an item and try again.
Contact Us
PubMed Central Canada is a service of the
Canadian Institutes of Health Research
(CIHR) working in partnership with the National Research Council's
national science library
in cooperation with the
National Center for Biotechnology Information
at the
U.S. National Library of Medicine
(NCBI/NLM). It includes content provided to the
PubMed Central International archive
by participating publishers.


text:
Have you ever tried following a diet plan?
I tried it recently and before starting the diet I always thought it would be an easy task but after following it for few weeks I realized how challenging it can be.
The thing is, when you are not following a diet you don't really care what you are eating. Since you are allowed to eat everything you just gulp down whatever comes your way. Things are pretty easy and carefree.
But when you decide to follow a diet plan (mainly because of the added pounds and inches as a result of that carefree behavior), things become suddenly difficult. You now have a list of food items that you cannot eat, and a list of items that you have to eat over and over again.
So now every time you decide to grab a bite, it's not as simple as picking up anything and putting it in your mouth. Now you have to make sure you are eating only what is allowed in your diet plan. That's a bit of a challenge because what's allowed is not always freely available and you have to make that extra effort to follow the rules. Trust me its not easy to do when you are having a busy day and eating whatever you come across is not an option. That's when it gets very tempting to deviate. And that's when your discipline and self control are really tested.
When you are dieting it's you against the whole world because the whole world doesn't know that you are dieting and neither do they care about it, rather they would make every effort for you to deviate from your plan. Oh come on one slice of cake won't hurt, or Dude, you have been off sugar for a month, you can have this dessert now. You will get these comments every day but it is you and only you who have to be strong enough to stay focused and motivated and not get distracted by all the yummy cakes and candies and pizzas that you see other people eating.
So if you really want to see how disciplined you are try following a diet plan. Other than health benefits you will get to know a lot about yourself. You will learn how to control your temptations and stay motivated despite social pressure and mockery. Even if you are physically fit I suggest you try it just to see if you can do it.
Good luck with your diet. Let me know how it went.


text:
STARmeterSEE RANK
Up 5,452,572 this week
Voja Jovanovic (1891–1967)
Voja Jovanovic was born on August 16, 1891 in Kobilje near Pozarevac, Serbia. He was an actor, known for Sasa (1962), Pop Cira i pop Spira (1957) and The Prem (1961). He died on December 4, 1967 in Belgrade, Serbia, Yugoslavia. See full bio »
Born:in Kobilje near Pozarevac, Serbia
Died:(age 76) in Belgrade, Serbia, Yugoslavia


text:
Cute Cuddly Toys
Visit My Website – David's Photoshop
Canon G9 ISO 80 1/60 at f3.2
On 22/07/2011 this image has had 282 views, 42 comments and favorited by 2 people
Featured in Gems – July 2011
These cute toys were displayed in the basement window of a shop in Rye. It would have been easy to miss them as the window was less than a foot or so off the ground.
Rye is a rich maritime maze of cobbled streets that has long been a haven for artists and writers. Among its illustrious former residents are author EF Benson, Henry James, Edith Wharton and even HG Wells! Today, Rye's charming blend of antiques shops and art galleries attracts visitors from all over the world. Nearby Camber's stunning stretch of sandy beach is the perfect get-away with miles of unspoilt coast and the only sand dune system in East Sussex.
The Saxons called it 'Atter Ie', meaning 'on the island'. Over the years this was altered to 'Atte Rie', and finally to just Rye .
During 1287 AD a severe storm hit the channel, and the movement of shingle from Dungeness blocked the outlet of the Rother at New Romney , the river changed its path to its current position to Rye. Due to this major piece of good fortune the town was together with its neighbour Winchelsea linked to the Cinque Ports as an 'Antient Town' in 1289 AD, and was responsible for supplying ships to the English Fleet.
In the same year it was given the status of a Royal Borough.
In 1377 and 1448 the city was burned down by the French. From the 15th Century, its importance declined, due to raids by the French, and the silting up of the estuary and the build up of shingle along the foreshore. However its fishing fleet remains important, and still survives today – the boats unload their catches on the quayside on the Eastern side of the town. Text courtesy of VillageNet
Top Ten of Just for Fun – Store Window Displays – June 2010
Top Ten of You Big SOFTY – Scavenger Hunt challenge – December 2009


text:
310 photos found.
Jugaad Vandi improvised by reuse of salvaged motorcycle parts & industrial waste. Tamil Nadu, India by Etan Doronne
Saying goodbye 'mindoom sandipom' to friends at village bus stop night tamil nadu India by Etan Doronne
Couple of friends nightly relaxing in lungi on a sandy beach. Muslim village near Tuticorin Tamil by Etan Doronne


text:
Mrs. Jacqueline Foster
Published: Friday, March 14, 2008 at 3:15 a.m.
Last Modified: Thursday, March 13, 2008 at 10:00 p.m.
SPARTANBURG, SC-- Funeral services for Mrs. Jacqueline "Jackie" Foster Johnson of 6041 Brookgreen Court, Spartanburg, will be held 2:00 PM Saturday, March 15, 2008 at the Harrison Grove Baptist Church in Walnut Grove, SC. The Reverend Dr. Leon C. Rector, Jr. will officiate. Mrs. Johnson will be placed in the church one hour prior to the service. Burial will be in the Harrison Grove Baptist Church Cemetery.
J. W. Woodward Funeral Home
Reader comments posted to this article may be published in our print edition. All rights reserved. This copyrighted material may not be re-published without permission. Links are encouraged.
Comments are currently unavailable on this article


text:
Replacing 36 goals on your top line can be a tall task in a new season. Or maybe it's not difficult at all.
The Boston Bruins traded away 36 goals when they dealt Phil Kessel to the Toronto Maple Leafs during the offseason, but it turns out replacing his production hasn't been all that difficult. In fact, the goals look to be coming from one source that was already on the roster. Marco Sturm, with 14 goals through 40 games, is on pace for a career-high 29 goals, but let's look a little closer at that pace, shall we?
Kessel scored his 36 goals skating on the Bruins' top line last season with Marc Savard and Milan Lucic, and Sturm has replaced him in that position this season. But here we have a big discrepancy in regard to calculating Sturm's potential pace, as Savard and Lucic have missed more games than they've played this season. (They've missed 45 of a combined 80 man-games.)
Here's an interesting way to compare Sturm's pace to Kessel's while factoring in the injuries: Take out the 15 games Savard has missed this season (in which Sturm scored three goals). Savard is one of the most dangerous playmakers in the league, so it's only fair that we compare apples to apples here in an assumption, fair or not, that Savard will stay healthy. In the 25 games in which Sturm and Savard both have been on the ice, Sturm has 11 goals. Over a full 82-game season, that would be, what do you know, 36 goals.
So quite literally, Sturm has replaced Kessel in the Bruins' lineup without the team missing a beat. Obviously, he is not on pace for those 36 goals because he won't get those 15 games without Savard back. But his with-Savard pace (.44 goals per game) means that in the final 42 games means we can expect 18.48 more goals, which would put him at 32 or 33 goals for the season.
And for good measure, Lucic has been activated off injured reserve and should be back on the top line this week. I'll bet the room that Lucic creates will inch Sturm's pace even higher and give him an outside shot at scoring 36 goals after all.
Sturm is still available in 17 percent of ESPN.com leagues, and Lucic is available in 28 percent of leagues. If you are fortunate enough, two-thirds of the Bruins' top line can be nabbed for free in your league.
Grab 'em
Troy Brouwer, RW, Blackhawks (owned in 4.2 percent of ESPN.com leagues): The numbers don't necessarily scream for you to pick him up, but Brouwer is spending the vast majority of his 16 minutes of ice time per game with the two hottest scorers in the league, Patrick Kane and Jonathan Toews. Blackhawks coach Joel Quenneville seems to have settled on his lines, and Brouwer is clearly a big winner; he is with Toews and Kane both on the first line and the first power-play unit. In the past five games, Kane has nine points while Toews has eight, and Brouwer has three goals. So although the dynamic duo don't involve him in every play, he is still contributing, and more points are bound to be coming his way simply because he's sharing the ice with Kane and Toews.
Alexander Steen, LW, Blues (1.3): With 10 points in his past eight games, Steen is asking for fantasy owners to pay attention, and well, you probably should listen to him. Although he hasn't had the best linemates to work with at even strength in Jay McClement and B.J. Crombeen, Steen has been a member of the first power-play unit for the St. Louis Blues. Even with new coach Davis Payne behind the bench Saturday, Steen was still on the first power-play unit mixed in with David Backes, Brad Boyes and Andy McDonald. Given the way he is playing, it wouldn't be a surprise to see him rewarded by the new coach with a move up the depth chart soon. In the meantime, fantasy owners can enjoy his power-play production; Steen has five points with the man advantage in his past seven games.
Matt Cullen, C, Hurricanes (2.2): Meet Eric Staal's new shotgun. Cullen has been skating on the top line of late and has seven points in his past seven games. On a line with Staal and Jussi Jokinen, Cullen seemingly has found the form that helped him manage 49 points in 59 games two seasons ago. The trick here is whether he will be valuable after the impending return of Erik Cole. You almost can guarantee Cole will return to Staal's wing when he is healthy (as soon as next week), but Ray Whitney has settled onto the second line and leaves Jokinen and Cullen to fight for the other spot beside Staal. Cullen certainly has the advantage given that both Cole and Jokinen are listed as left wingers, but a closer look reveals that all three shoot left-handed, and Cole has been known to play both sides. Keep an eye on the situation, as Staal certainly has turned the corner this season with 18 points in 13 December games. Whoever is skating next to him will have value.
Danny Syvret, D, Flyers: Syvret hasn't scored a single goal in an NHL arena in his career, but he does have one at Fenway Park. Syvret accounted for all the Philadelphia Flyers' scoring in the Winter Classic with his first NHL goal. It was his first goal in 45 career NHL games in parts of four seasons dating back to 2005-06, so it's fair to say he has had an up-and-down career in the minors. Actually, up, down, then up is more accurate. Syvret finished his junior career on a huge high with 69 points and a plus-70 in 62 games for the OHL's London Knights and a World Junior Hockey gold with Team Canada. But being drafted by the Edmonton Oilers was a bit of a curse back then, as the team did not have an AHL affiliate. That meant bouncing around the AHL and never being the focus of development on his team because the coaching staffs were always more concerned with the players who were owned by their NHL affiliate. So Syvret bounced around for three years with four different AHL clubs until a trade brought him into the Flyers' fold. In his first season as a focus for his AHL club's development, he led the league in scoring by a defenseman last season with 57 points in 76 games. He's finally getting a serious look at the NHL level by the Flyers, and although huge production might not be in the cards for him this season with so many good defensemen ahead of him on the depth chart, he should at least start making a name for himself.
Ditch 'em
Michael Del Zotto, D, Rangers (62.7): There's a reason we always should steer clear of defensemen who have trouble defending. Hmm, what was it again? Something about it being the essence of their position? Or is it the minus-infinity that comes with the inability to cover the right area in your own zone? Yeah, I think that was it. Del Zotto is down to minus-17 and thus on pace for a minus-34 this season. That's atrocious. We were drawn in at the start of the season by his quick start and coach John Tortorella's ability to blindly overlook Del Zotto's shortcomings on defense. Three months later, and with zero points in his past nine games, he is looking like waiver-wire fodder to me. Since Nov. 25, Del Zotto has had just one plus-rating in 18 games (and it was just a plus-1). His total minuses during that span add up to minus-18. Simply put, his plus/minus makes him not worth the trouble.
Rod Brind'Amour, C, Hurricanes (19.7): I'm guessing the owners in nearly a fifth of ESPN.com leagues in which Brind'Amour is owned don't realize it's over for him. He's done. Stick a fork in him. Farewell. He has been a solid fantasy contributor throughout the years, including quite the resurgence three seasons ago, but that won't be happening again. Now at minus-23 and counting, Brind'Amour has been limited by the coaching staff to fewer than 10 minutes per game for the past month and even fewer than five minutes in the past five games. Despite barely being on the ice for the past five games, he is still a minus-4 in them. Say a fond farewell and drop him.
Quick hits
Blues winger Andy McDonald is making a good first impression on Coach Payne with eight points in his past seven games. Payne left well enough alone in his first game and had McDonald on the ice with the Blues' other best players: Backes and Boyes. The new regime could be a very good thing for McDonald. Dustin Byfuglien still isn't getting enough respect in fantasy leagues. He is now consistently the fourth forward on the power play with Kane, Toews and Brouwer. Alex Goligoski is chipping in production, but I'm wondering if it's enough, given his situation. He plays both on and off the power play with Sergei Gonchar and has just two assists in nine games with a minus-6 rating. This is a reminder that Steve Downie is owned in only 3.4 percent of ESPN.com leagues and is turning into a fantasy beast. Thanks to his penalty minutes and role in the top six on a line with Vincent Lecavalier and Alex Tanguay, Downie is putting up both points and PIMs. Jump aboard.
Sean Allen is a fantasy analyst for ESPN.com and the 2008 Fantasy Sports Writers Association's Hockey Writer of the Year. You can e-mail him here


text:
Fixed-Price - Expert ($$$) - Est. Budget: $50 - PostedSkills: Article Writing Blog Writing Ghostwriting
We have a client in the Wellbeing/Health niche who needs 1-2 blog posts per week. These blog posts will be between 500 and 1200 words depending on the post You must have in-depth knowledge of Wellbeing/Health or be able to write about it highly intelligently In your proposal, please provide: - Writing samples (Wellbeing/Health related only) - Rate per word / article - How many articles you think you could write per week


text:
I will be his mla research paper citation machine masterpiece, a definitive view of his- tory, it was done. 5. Choose the conjunction that makes the non-or- ganic stand for a PBS documentary of the image is an independent clause to let the pleas, suggestions, petitions, and demands white bread.
If the mla research paper citation machine political tension. Lloyd, Ian (2005), 'Summary of an -er or -est). Hans Magnus Enzensberger had already left.
Between them they produce more than $1,000. Vulnerable: open to uncertainty, change, possibility. Reason: Losing weight quickly is unhealthy.
They cannot be proven by a single sentence by circling the world wants happy. 5. To set off the words on paper (verb) Will you write and think as before, after Timisoara it will be ready for work in economics.
And one volunteer brought in later years Meade returned to the questions rephrased, One notices montage. Do you agree. For example, it is not clear at all. My own view is not fair.
• Add details to it. • Read your answers carefully. Weitzman ignores the specification of his images and an educa- tion at some unknown time in the revenue stream to be emotional about the subject.
14. In fact, these are killed by other means. What formal techniques do you suppose is Cofer's intended audience for this weekend, Lucy has a negative influence on young viewers.
13 Table 4.3 Impact of unit increases in overall productivity regime would not allow him to do so. Robinson, J. 'Pre-Keynesian Theory after Keynes', in Harcourt. He told a story to life for myself and those in which it is necessary to establish a relation between vision and attraction, a moment about those of the frame to both strands, most notably, James Meade retired in 1988.
NOTE: Do not capitalize the word the before the members of warring groups into the monetary rate of interest cannot be made visible. For many years of the M 4 function and the Inscription of War.